Pharmacological Activation of Nrf2 Enhances Functional Liver Regeneration.

BACKGROUND AND AIMS: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an array of cytoprotective genes, yet studies in transgenic mice have led to conflicting reports on its role in liver regeneration. We aimed to test the hypothesis that pharmacological activation of Nrf2 would enhance liver regeneration. APPROACH AND RESULTS: Wild-type and Nrf2 null mice were administered bardoxolone methyl (CDDO-Me), a potent activator of Nrf2 that has entered clinical development, and then subjected to two-thirds partial hepatectomy. Using translational noninvasive imaging techniques, CDDO-Me was shown to enhance the rate of restoration of liver volume (MRI) and improve liver function (multispectral optoacoustic imaging of indocyanine green clearance) in wild-type, but not Nrf2 null, mice following partial hepatectomy. Using immunofluorescence imaging and whole transcriptome analysis, these effects were found to be associated with an increase in hepatocyte hypertrophy and proliferation, the suppression of immune and inflammatory signals, and metabolic adaptation in the remnant liver tissue. Similar processes were modulated following exposure of primary human hepatocytes to CDDO-Me, highlighting the potential relevance of our findings to patients. CONCLUSIONS: Our results indicate that pharmacological activation of Nrf2 is a promising strategy for enhancing functional liver regeneration. Such an approach could therefore aid the recovery of patients undergoing liver surgery and support the treatment of acute and chronic liver disease.

Exosomal Transport of Hepatocyte-Derived Drug-Modified Proteins to the Immune System.

Idiosyncratic drug-induced liver injury (DILI) is a rare, often difficult-to-predict adverse reaction with complex pathomechanisms. However, it is now evident that certain forms of DILI are immune-mediated and may involve the activation of drug-specific T cells. Exosomes are cell-derived vesicles that carry RNA, lipids, and protein cargo from their cell of origin to distant cells, and they may play a role in immune activation. Herein, primary human hepatocytes were treated with drugs associated with a high incidence of DILI (flucloxacillin, amoxicillin, isoniazid, and nitroso-sulfamethoxazole) to characterize the proteins packaged within exosomes that are subsequently transported to dendritic cells for processing. Exosomes measured between 50 and 100 nm and expressed enriched CD63. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) identified 2,109 proteins, with 608 proteins being quantified across all exosome samples. Data are available through ProteomeXchange with identifier PXD010760. Analysis of gene ontologies revealed that exosomes mirrored whole human liver tissue in terms of the families of proteins present, regardless of drug treatment. However, exosomes from nitroso-sulfamethoxazole-treated hepatocytes selectively packaged a specific subset of proteins. LC/MS-MS also revealed the presence of hepatocyte-derived exosomal proteins covalently modified with amoxicillin, flucloxacillin, and nitroso-sulfamethoxazole. Uptake of exosomes by monocyte-derived dendritic cells occurred silently, mainly through phagocytosis, and was inhibited by latrunculin A. An amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SRY (sex determining region Y)-box 30 activated naïve T cells from human leukocyte antigen A*02:01-positive human donors. Conclusion: This study shows that exosomes have the potential to transmit drug-specific hepatocyte-derived signals to the immune system and provide a pathway for the induction of drug hapten-specific T-cell responses.

Dynamic and accurate assessment of acetaminophen-induced hepatotoxicity by integrated photoacoustic imaging and mechanistic biomarkers in vivo.

The prediction and understanding of acetaminophen (APAP)-induced liver injury (APAP-ILI) and the response to therapeutic interventions is complex. This is due in part to sensitivity and specificity limitations of currently used assessment techniques. Here we sought to determine the utility of integrating translational non-invasive photoacoustic imaging of liver function with mechanistic circulating biomarkers of hepatotoxicity with histological assessment to facilitate the more accurate and precise characterization of APAP-ILI and the efficacy of therapeutic intervention. Perturbation of liver function and cellular viability was assessed in C57BL/6J male mice by Indocyanine green (ICG) clearance (Multispectral Optoacoustic Tomography (MSOT)) and by measurement of mechanistic (miR-122, HMGB1) and established (ALT, bilirubin) circulating biomarkers in response to the acetaminophen and its treatment with acetylcysteine (NAC) in vivo. We utilised a 60% partial hepatectomy model as a situation of defined hepatic functional mass loss to compared acetaminophen-induced changes to. Integration of these mechanistic markers correlated with histological features of APAP hepatotoxicity in a time-dependent manner. They accurately reflected the onset and recovery from hepatotoxicity compared to traditional biomarkers and also reported the efficacy of NAC with high sensitivity. ICG clearance kinetics correlated with histological scores for acute liver damage for APAP (i.e. 3h timepoint; r=0.90, P<0.0001) and elevations in both of the mechanistic biomarkers, miR-122 (e.g. 6h timepoint; r=0.70, P=0.005) and HMGB1 (e.g. 6h timepoint; r=0.56, P=0.04). For the first time we report the utility of this non-invasive longitudinal imaging approach to provide direct visualisation of the liver function coupled with mechanistic biomarkers, in the same animal, allowing the investigation of the toxicological and pharmacological aspects of APAP-ILI and hepatic regeneration.

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.

The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems.

A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity.

Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.

Total proteome analysis identifies migration defects as a major pathogenetic factor in immunoglobulin heavy chain variable region (IGHV)-unmutated chronic lymphocytic leukemia.

The mutational status of the immunoglobulin heavy chain variable region defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and unmutated (UM-CLL). To elucidate the molecular mechanisms underlying the adverse clinical outcome associated with UM-CLL, total proteomes from nine UM-CLL and nine M-CLL samples were analyzed by isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry. Based on the expression of 3521 identified proteins, principal component analysis separated CLL samples into two groups corresponding to immunoglobulin heavy chain variable region mutational status. Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells underexpressed proteins associated with cytoskeletal remodeling and overexpressed proteins associated with transcriptional and translational activity. Taken together, our findings indicate that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes, where they are exposed to proliferative stimuli. In keeping with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed a strong and specific association between UM-CLL and lymphadenopathy. Our study illustrates the potential of total proteome analysis to elucidate pathogenetic mechanisms in cancer.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney.

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 h, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared with those of wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared with those of vehicle control. In the light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis, and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

BACKGROUND & AIMS: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. METHODS: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. RESULTS: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. CONCLUSIONS: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes.

Proteome-wide analyses of human hepatocytes during differentiation and dedifferentiation.

UNLABELLED: Failure to predict hepatotoxic drugs in preclinical testing makes it imperative to develop better liver models with a stable phenotype in culture. Stem cell-derived models offer promise, with differentiated hepatocyte-like cells currently considered to be "fetal-like" in their maturity. However, this judgment is based on limited biomarkers or transcripts and lacks the required proteomic datasets that directly compare fetal and adult hepatocytes. Here, we quantitatively compare the proteomes of human fetal liver, adult hepatocytes, and the HepG2 cell line. In addition, we investigate the proteome changes in human fetal and adult hepatocytes when cultured in a new air-liquid interface format compared to conventional submerged extracellular matrix sandwich culture. From albumin and urea secretion, and luciferase-based cytochrome P450 activity, adult hepatocytes were viable in either culture model over 2 weeks. The function of fetal cells was better maintained in the air-liquid interface system. Strikingly, the proteome was qualitatively similar across all samples but hierarchical clustering showed that each sample type had a distinct quantitative profile. HepG2 cells more closely resembled fetal than adult hepatocytes. Furthermore, clustering showed that primary adult hepatocytes cultured at the air-liquid interface retained a proteome that more closely mimicked their fresh counterparts than conventional culture, which acquired myofibroblast features. Principal component analysis extended these findings and identified a simple set of proteins, including cytochrome P450 2A6, glutathione S transferase P, and alcohol dehydrogenases as specialized indicators of hepatocyte differentiation. CONCLUSION: Our quantitative datasets are the first that directly compare multiple human liver cells, define a model for enhanced maintenance of the hepatocyte proteome in culture, and provide a new protein "toolkit" for determining human hepatocyte maturity in cultured cells.

The generation, detection, and effects of reactive drug metabolites.

The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.

Stem cell-derived hepatocytes as a predictive model for drug-induced liver injury: are we there yet?

Amongst the different types of adverse drug reactions, drug-induced liver injury is the most prominent cause of patient morbidity and mortality. However, the current available hepatic model systems developed for evaluating safety have limited utility and relevance as they do not fully recapitulate a fully functional hepatocyte, and do not sufficiently represent the genetic polymorphisms present in the population. The rapidly advancing research in stem cells raises the possibility of using human pluripotent stem cells in bridging this gap. The generation of human induced pluripotent stem cells via reprogramming of mature human somatic cells may also allow for disease modelling in vitro for the purposes of assessing drug safety and toxicology. This would also allow for better understanding of disease processes and thus facilitate in the potential identification of novel therapeutic targets. This review will focus on the current state of effort to derive hepatocytes from human pluripotent stem cells for potential use in hepatotoxicity evaluation and aims to provide an insight as to where the future of the field may lie.

Evidence for metabolic cleavage of a PEGylated protein in vivo using multiple analytical methodologies.

PEGylation of therapeutic proteins is commonly used to extend half-lives and to reduce immunogenicity. However, reports of antibodies toward PEGylated proteins and of poly(ethylene glycol) (PEG) accumulation suggest that efficacy and safety concerns may arise. To understand the relationship among the pharmacology, immunogenicity, and toxicology of PEGylated proteins, we require knowledge of the disposition and metabolic fate of both the drug and the polymer moieties. The analysis of PEG by standard spectrophotometric or mass spectrometric techniques is problematic. Consequently, we have examined and compared two independent analytical approaches, based on gel electrophoresis and nuclear magnetic resonance (NMR) spectroscopy, to determine the biological fate of a model PEGylated protein, (40K)PEG-insulin, within a rat model. Both immunoblotting with an antibody to PEG and NMR analyses (LOD 0.5 µg/mL for both assays) indicated that the PEG moiety remained detectable for several weeks in both serum and urine following intravenous administration of (40K)PEG-insulin (4 mg/kg). In contrast, Western blotting with anti-insulin IgG indicated that the terminal half-life of the insulin moiety was far shorter than that of the PEG, providing clear evidence of conjugate cleavage. The application of combined analytical techniques in this way thus allows simultaneous independent monitoring of both protein and polymer elements of a PEGylated molecule. These methodologies also provide direct evidence for cleavage and definition of the chemical species present in biological fluids which may have toxicological consequences due to unconjugated PEG accumulation or immunogenic recognition of the uncoupled protein.

Microwave ablation of ex vivo human liver and colorectal liver metastases with a novel 14.5 GHz generator.

PURPOSE: This study assessed the relationship between time, power and ablation size using a novel high-frequency 14.5 GHz microwave applicator in ex vivo human hepatic parenchyma and colorectal liver metastases. Previous examination has demonstrated structurally normal but non-viable cells within the ablation zone. This study aimed to further investigate how ablation affects these cells, and to confirm non-viability. MATERIALS AND METHODS: Ablations were performed in ex vivo human hepatic parenchyma and tumour for a variety of time (10-180 s) and power (10-50 W) settings. Histological examination was performed to assess cellular anatomy, whilst enzyme histochemistry was used to confirm cellular non-viability. Transmission electron microscopy was used to investigate the subcellular structural effects of ablation within these fixed cells. Preliminary proteomic analysis was also performed to explore the mechanism of microwave cell death. RESULTS: Increasing time and power settings led to a predictable and reproducible increase in size of ablation. At 50 W and 180 s application, a maximum ablation diameter of 38.8 mm (±1.3) was produced. Ablations were produced rapidly, and at all time and power settings ablations remained spherical (longest:shortest diameter <1.2). Routine histological analysis using haematoxylin-eosin (H&E) confirmed well preserved cellular anatomy despite ablation. Transmission electron microscopy demonstrated marked subcellular damage. Enzyme histochemistry showed complete absence of viability in ablated tissue. CONCLUSIONS: Large spherical ablation zones can be rapidly and reproducibly achieved in ex vivo human hepatic parenchyma and colorectal liver metastases using a 14.5 GHz microwave generator. Despite well preserved cellular appearance, ablated tissue is non-viable.

Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway.

Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy.

BACKGROUND: Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer. RESULTS: Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001). CONCLUSIONS: Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.

Assessing technical and biological variation in SWATH-MS-based proteomic analysis of chronic lymphocytic leukaemia cells.

Chronic lymphocytic leukaemia (CLL) exhibits variable clinical course and response to therapy, but the molecular basis of this variability remains incompletely understood. Data independent acquisition (DIA)-MS technologies, such as SWATH (Sequential Windowed Acquisition of all THeoretical fragments), provide an opportunity to study the pathophysiology of CLL at the proteome level. Here, a CLL-specific spectral library (7736 proteins) is described alongside an analysis of sample replication and data handling requirements for quantitative SWATH-MS analysis of clinical samples. The analysis was performed on 6 CLL samples, incorporating biological (IGHV mutational status), sample preparation and MS technical replicates. Quantitative information was obtained for 5169 proteins across 54 SWATH-MS acquisitions: the sources of variation and different computational approaches for batch correction were assessed. Functional enrichment analysis of proteins associated with IGHV mutational status showed significant overlap with previous studies based on gene expression profiling. Finally, an approach to perform statistical power analysis in proteomics studies was implemented. This study provides a valuable resource for researchers working on the proteomics of CLL. It also establishes a sound framework for the design of sufficiently powered clinical proteomics studies. Indeed, this study shows that it is possible to derive biologically plausible hypotheses from a relatively small dataset.

Network analysis of primary hepatocyte dedifferentiation using a shotgun proteomics approach.

The liver is the major site of xenobiotic metabolism and detoxification. Primary cultures of hepatocytes are a vital tool in the development of new therapeutic agents but their utility is hindered by the rapid loss of phenotype. Hepatocytes cultured in a sandwich of extracellular matrix protein maintain better hepatic function compared with cells cultured as a monolayer but a wide-ranging proteomics study of the differences in cultures has never been performed. We characterize the changing phenotype of rat hepatocytes in primary culture using iTRAQ proteomics and systems biology network analysis of the identified, significantly regulated, proteins. A total of 754 unique proteins were identified from 4 independent experiments. Of these, 413 proteins were common to at least 3 experiments and 328 proteins were identified in all experiments. Both culture systems displayed altered expression of many common proteins. Network analysis showed that the primary functions of these proteins were in metabolic pathways, immune responses and cytoskeleton remodelling. Monolayer cultures uniquely regulate proteins mapping to pathways of oxidative stress and cell migration, whereas sandwich culture affected translation regulation and apoptosis pathways. These experiments provide a detailed proteomics data set to direct further work into maintaining hepatic phenotype using cultured primary hepatocytes and stem cell derived hepatocyte-like cells.

The keap1-nrf2 cellular defense pathway: mechanisms of regulation and role in protection against drug-induced toxicity.

Adverse drug reactions pose a significant public health problem. In some cases, the process of drug metabolism can contribute to the onset of toxicity through the bioactivation of a parent molecule to a chemically reactive intermediate. In order to maintain a favorable balance between bioactivation and detoxification, mammalian cells have evolved an inducible cell defense system known as the antioxidant response pathway. The activity of this cytoprotective pathway is largely regulated by the transcription factor Nrf2, which governs the expression of many phase II detoxification and antioxidant enzymes. In turn, the activity of Nrf2 is regulated by the cysteine-rich cytosolic inhibitor Keap1, which acts as a "sensor" for chemical/oxidative stress. This article summarizes our current understanding of the molecular mechanisms that regulate the function of the Keap1-Nrf2 pathway and highlights the importance of Nrf2 in the protection against drug-induced toxicity.

The hepatotoxic metabolite of acetaminophen directly activates the Keap1-Nrf2 cell defense system.

UNLABELLED: The transcription factor Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. We have demonstrated previously that acetaminophen activates Nrf2 in mouse liver following administration of non-hepatotoxic and hepatotoxic doses in vivo, implying that Nrf2 may have an important role in the protection against drug-induced liver injury. Nrf2 activation has been proposed to occur through the modification of cysteine residues within Keap1, the cytosolic repressor of Nrf2. We hypothesized that acetaminophen activates Nrf2 via the formation of its reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which may disrupt the repression of Nrf2 through the modification of cysteine residues within Keap1. Here, we show that NAPQI can directly activate the Nrf2 pathway in mouse liver cells, inducing an adaptive defense response that is antagonized by RNA interference targeted against Nrf2. Furthermore, mass spectrometric analysis shows that NAPQI selectively modifies cysteine residues in Keap1, both in recombinant protein in vitro and in cells ectopically expressing Keap1. Using this cell-based model, we demonstrate that activation of Nrf2 by NAPQI and a panel of probe molecules [dexamethasone 21-mesylate, 15-deoxy-Delta-((12,14))-prostaglandin J(2), 2,4-dinitrochlorobenzene, and iodoacetamide] correlates with the selective modification of cysteine residues located within the intervening region of Keap1. However, substantial depletion of glutathione (to less than 15% of basal levels) by buthionine sulfoximine, which does not directly modify Keap1, is also sufficient to activate Nrf2. CONCLUSION: Nrf2 can be activated via the direct modification of cysteine residues located within the intervening region of Keap1, but also via the substantial depletion of glutathione without the requirement for direct modification of Keap1. It is possible that both of these mechanisms contribute to the activation of Nrf2 by acetaminophen.

A mechanistic investigation into the irreversible protein binding and antigenicity of p-phenylenediamine.

Exposure to the skin sensitizer p-phenylenediamine (PPD) is associated with allergic contact dermatitis; however, the ability of PPD to modify protein has not been fully investigated. The aims of this study were to characterize the reactions of PPD and the structurally related chemical 2,5-dimethyl-1,4-benzoquinonediamine with model nucleophiles, a synthetic peptide (DS3) containing each of the naturally occurring amino acids and His-tagged glutathione-S-transferase pi (GSTP), and to explore the effect of dimethyl substitution on PPD-specific T-cell responses using lymphocytes from allergic patients. The reductive soft nucleophiles N-acetyl cysteine and glutathione prevented PPD self-conjugation reactions and Bandrowski's base formation, but no adducts were detected. N-Acetyl lysine, a hard nucleophile, did not alter the rate of PPD degradation or form PPD adducts. With PPD and 2,5-dimethyl-1,4-benzoquinonediamine, only cysteine was targeted in the DS3 peptide. PPD and 2,5-dimethyl-1,4-benzoquinonediamine were also found to selectively modify the reactive Cys 47 residue of GSTP, which has a pK(a) of 3.5-4.2 and therefore exists in a largely protonated form. Glutathione formed mixed disulfides with the DS3 peptide, reducing levels of PPD binding. Lymphocytes from PPD allergic patients proliferated in the presence of PPD but not with 2,5-dimethyl-1,4-benzoquinonediamine. These results reveal that PPD and 2,5-dimethyl-1,4-benzoquinonediamine bind selectively to specific cysteine residues in peptides and proteins. Lymphocytes from PPD allergic patients were capable of discriminating between the different haptenic structures, suggesting that the hapten, but not the peptide moiety associated with MHC, is an important determinant for T-cell recognition.